The long-term goal of this research is to comprehensively understand the mechanisms and regulation of intracellular protein. Protein degradation regulates nearly every aspect of normal cellular function; dysfunction of protein degradation underlies many diseases including cancer, muscle wasting, and neuropathologies. Most protein degradation in eukaryotic cells is catalyzed by the 26S proteasome, the ATP-dependent protease of the ubiquitin system. The goal of this project is to determine the mechanisms by which ATP binding and hydrolysis mediate proteasome function, using biochemically-defined in vitro systems. Specific Aim 1 will characterize biochemical features of ATP binding and hydrolysis by the 26S proteasome and define the relative qualitative and quantitative contributions of six different AAA ATPase subunits to these processes. These experiments will support subsequent mechanistic studies on the roles of ATP and the AAA subunits in proteasome function. Specific Aim 2 will determine molecular mechanisms by which ATP binding mediates assembly of the 26S proteasome from protease (20S proteasome) and regulatory (PA700/19S) subcomplexes, and activation of protease activity upon assembly. These experiments will test the hypothesis that proteasome assembly and activation require separate ATP binding events and define roles or individual AAA subunits in these processes. These experiments will distinguish between alternative models in which AAA subunits have distinct/dedicated roles or multiple/redundant roles. Specific Aim 3 will define the role of ATP hydrolysis in 26S proteasome-catalyzed protein degradation. These experiments will compare relative rates of degradation of variants of a model substrate that differ systematically in structural stability and proteasome targeting elements, such as structurally defined polyubiquitin chains. By comparing the qualitative and quantitative requirements for ATP hydrolysis and proteolysis, these experiments will deconvolute the roles of ATP consumption in multiple subfunctions of proteolysis including, substrate binding, unfolding, translocation, and deubiquitylation. Drugs that inhibit the proteasome are used to treat cancer and are in clinical trials for other diseases. Thus, elucidation of fundamental molecular mechanisms of proteasome func-tion will provide the basis for development of better antiproteasome drugs to treat human disease.